Thrombosis involves cell adhesion of platelets. Platelet cell adhesion generally involves recognition of specific adhesive proteins by the platelet cell surface receptor GPIIb-IIIa.
GPIIb-IIIa is a noncovalent, Ca++ dependent, heterodimer complex comprised of noncovalently associated alpha and beta subunits. Jennings et al., J. Biol. Chem., 257:10458–10466 (1982). The alpha subunit, GPIIb consists of a heavy chain (hGPIIb) having a relative molecular weight of about 120 kilodaltons (KDa), and a light chain (lGPIIb) of about 20 KDa that are linked together by disulfide bonds. The beta subunit, GPIIIa is a single chain polypeptide of about 100 KDa. Phillips et al., J. Biol. Chem., 252:2121–2126 (1977).
GPIIb-IIIa contributes to platelet function through interactions with RGD-containing proteins such as fibrinogen [Bennett et al., Proc. Natl. Acad. Sci. USA, 80:2417–2421 (1983)], fibronectin [Ginsberg et al., J. Clin. Invest., 71:619–624 (1983)], and von Willebrand factor [Ruggeri et al., Proc. Natl. Acad. Sci. USA, 79:6038–6041 (1982)], and therefore is a component of the common platelet adhesive protein receptor [Pytela et al., Science, 231:1559–1562 (1986) and Plow et al., J. Biol. Chem., 259:5388–5391 (1984)].
The sites on GPIIb-IIIa that are required to function as an adhesion receptor are not well characterized. Several observations suggest that a functionally significant site on GPIIb-IIIa is near the epitope defined by the monoclonal antibody PMI-1. This antibody binds to the heavy chain of GPIIb [Shadle et al., J. Cell. Biol., 99:2056–2060 (1984)] and defines a region of GPIIb that is associated with several distinct functional activities. First, PMI-1 inhibits adhesion of washed platelets to collagen. Shadle et al., J. Cell. Biol., 99:2056–2060 (1984). Second, the surface orientation of this region is regulated by divalent cations because millimolar (mM) concentrations of calcium or magnesium suppress expression of the PMI-1 epitope. Ginsberg et al., J. Clin. Invest., 78:1103–1111 (1986). Third, abnormal divalent cation regulation of the conformation of this site is associated with a functional thrombasthenic state. Ginsberg et al., J. Clin. Invest., 78:1103–1111 (1986). Fourth, stimulation of platelets with up to 100 micromolar adenosine diphosphate (ADP) or epinephrine, 1 unit per milliliter thrombin, or 50 micrograms per milliliter calf skin collagen does not substantially increase the binding of PMI-1 antibodies to platelets.
Platelet activation has been reported to produce the appearance of antigenic sites on the platelet surface that are not present in the non-activated platelet, and at least one of such induced sites has been localized to the GPIIb-IIIa receptor complex. Shattil et al, J. Biol. Chem. 260:11107–11114 (1985); Coller, B. S., J. Cell Biol., 103:451–456 (1986).
Numerous assays based on the release of fragments of blood coagulation enzymes or platelet activation have been developed in attempts to identify thrombotic and pre-thrombotic states. From the platelet perspective, initial efforts were focused on release of platelet specific antigens. Ginsberg, et al., In Immunologic Analysis: Recent Progress in Diagnostic Labor. Immunol., Nakamura et al., Eds. Masson Publishing, USA Inc., 199–209 (1982). Recent studies have focused on the use of monoclonal antibodies selective for activated platelets. Frelinger et al., J. Biol. Chem., 263:12397–12402 (1988); Aiken et al., Sem. Thromb. Hemost., 13:307–316 (1987); Berman et al., J. Clin. Invest., 78:130–137 (1986); Hsu-Lin et al., J. Biol. Chem., 259:9121–9126 (1984); George et al., J. Clin. Invest., 78:340–348 (1986); Shattil et al., Blood, 73:150–158 (1989); Shattil et al., J. Biol. Chem., 260:11107–11114 (1985); Adelman et al., Blood, 70:1362–1366 (1987).
Each of the above methods has been plagued by unintentional in vitro activation because it is difficult to obtain blood samples in a manner such that the platelet activation events are not triggered during venipuncture, sample handling or the like. This difficulty has severely limited the clinical utility of such assays. It would be desirable, therefore, to develop a method to detect the occurrence of thrombotic events without relying on platelet activation events. The utility of determining the elevation in a plasma glycocalicin, as a potential assay for in vivo platelet activation, has also been reported. Steinberg et al., N. Engl. J. Med., 317:1037–1042 (1987); Coller et al., J. Clin. Invest., 73:794–799 (1984).
It has recently been found that a class of antigenic determinants are expressed when GPIIb-IIIa specifically binds to its ligand. The antigenic determinants are not expressed by either the non-occupied GPIIb-IIIa receptor or the non-bound ligand. One such determinant located on the C-terminus of the GPIIB heavy chain is recognized by the previously described PMI-1 monoclonal antibody. Shadle et al., J. Cell Biol., 99:2056–2060 (1984); Frelinger et al., J. Biol. Chem., 263:12397–12402 (1988).